Revision Bulletin
Official December 1, 2014
Beta Carotene 1
Acceptance criteria: 96.0%–101.0% of total carotenoids as beta carotene (C40H56)
.
Beta Carotene
Change to read:
C40H56
536.87
β,β-Carotene;
all-trans-β-Carotene;
(all-E)-1,1′-(3,7,12,16-Tetramethyl-1,3,5,7,9,11,13,15,17octadecanonaene-1,18-diyl)bis[2,6,6-trimethylcyclohexene] [7235-40-7].
DEFINITION
Change to read:
Beta Carotene contains NLT 96.0% and NMT 101.0% of
total carotenoids calculated as beta carotene (C40H56). It
contains NLT •95%• (RB 1-Dec-2014) of all-trans-beta carotene
in the total carotenoids content.
.
•• (RB 1-Dec-2014)
.
IDENTIFICATION
• A.
Sample solution: Prepare as directed in the Sample solution in the test for Content of Total Carotenoids.
Analysis: Record the UV-Vis spectrum from 300–600
nm.
Acceptance criteria: The Sample solution shows a
shoulder at about 427 nm, an absorption maximum at
about 455 nm, and another maximum at about 483
nm. The absorbance ratio A455/A483 is between 1.14
and 1.18.
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the test for Content of Beta Carotene.
COMPOSITION
Change to read:
• CONTENT OF TOTAL CAROTENOIDS
[NOTE—Use low-actinic glassware.]
Sample stock solution: 0.1 mg/mL of Beta Carotene in
tetrahydrofuran
Sample solution: Transfer 3.0 mL of Sample stock solution to a 100-mL volumetric flask, and dilute with cyclohexane to volume.
Instrumental conditions
(See Spectrophotometry and Light-Scattering 〈851〉.)
Analytical wavelength: •456• (RB 1-Dec-2014) nm
Cell path: 1 cm
Blank: Cyclohexane
Analysis
Sample: Sample solution
Calculate the percentage of total carotenoids (T) as
beta carotene (C40H56):
.
T = A/(F × C)
A
F
= absorbance of the Sample solution
= 2505, coefficient of extinction (E1%) of pure
all-trans-beta carotene in cyclohexane
(100 mL · g−1 · cm−1)
= concentration of the Sample solution (g/mL)
.
.
C
.
• CONTENT OF BETA CAROTENE
[NOTE—Use low-actinic glassware.]
Mobile phase: Transfer 50 mg of butylated hydroxytoluene to a 1-L volumetric flask, and dissolve with 20 mL
of 2-propanol. Add 0.2 mL of N-ethyldiisopropylamine,
25 mL of 0.2% ammonium acetate solution, 455 mL of
acetonitrile, and about 450 mL of methanol. Allow the
solution to reach room temperature, and dilute with
methanol to volume.
Diluent: 50 µg/mL of butylated hydroxytoluene in
alcohol
System suitability solution: Transfer 20 mg of USP
Beta Carotene System Suitability RS to a 50-mL volumetric flask. Add 1 mL of water and 4 mL of tetrahydrofuran, and sonicate for 5 min. Dilute with Diluent to
volume, and sonicate for 5 min. Cool to room temperature, pass the suspension through a membrane filter
of 0.45-µm pore size, and use the clear filtrate.
Standard solution: 10 µg/mL of USP Beta Carotene RS
in tetrahydrofuran and Diluent (1:9). Dissolve an appropriate amount of USP Beta Carotene RS in a volumetric
flask first with tetrahydrofuran, using 10% of the volume of the flask, then dilute with Diluent to volume.
Sample solution: Dilute the freshly prepared Sample
stock solution as prepared in the test for Content of Total Carotenoids (1 in 10) with Diluent.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 448 nm
Column: 4.6-mm × 25-cm; 5-µm packing L68
Column temperature: 30°
Flow rate: 0.6 mL/min
Injection volume: 20 µL
System suitability
Samples: System suitability solution and Standard
solution
The approximate relative retention times of the components in the System suitability solution are listed in
Table 1.
Table 1
Analyte
all-trans-Alpha carotene
all-trans-Beta carotene
9-cis-Beta carotene
13-cis-Beta carotene
15-cis-Beta carotene
Relative
Retention
Time
0.93
1.00
1.07
1.17
1.21
Relative
Response
Factor
•1.0•
.
(RB 1-Dec-2014)
1.0
1.0
1.2
1.4
Suitability requirements
Chromatogram similarity: The chromatogram from
the System suitability solution is similar to the reference chromatogram provided with the lot of USP
Beta Carotene System Suitability RS being used.
Resolution: NLT 1.5 between all-trans-beta carotene
and all-trans-alpha carotene; •NLT 1.2• (RB 1-Dec-2014)
between all-trans-beta carotene and 9-cis-beta carotene, System suitability solution
Tailing factor: NMT 2.0 for the all-trans-beta carotene peak, Standard solution
2014 The United States Pharmacopeial Convention All Rights Reserved.
.
2
Revision Bulletin
Official December 1, 2014
Beta Carotene
= [(peak area of all-trans-alpha carotene ×
•1.0• (RB 1-Dec-2014)) + (peak area of all-transbeta carotene) + (peak area of 9-cis-beta
carotene) + (peak area of 13-cis-beta
carotene × 1.2) + (peak area of 15-cis-beta
carotene × 1.4) + (sum of peak areas of
other cis-isomers of beta carotene)] from the
Sample solution
Acceptance criteria
Alpha carotene: NMT 1.0%
Total related compounds (including alpha carotene):
NMT •5%• (RB 1-Dec-2014)
Relative standard deviation: NMT 2.0% for the alltrans-beta carotene peak from replicate injections,
Standard solution
Analysis
Sample: Sample solution
Record the chromatograms, and identify the peaks of
the relevant analytes of the Sample solution by comparing with those of the System suitability solution.
Measure the peak area responses.
Calculate the percentage of all-trans-beta carotene relative to total carotenoids in the sample taken:
Result = (rU/rT) × 100
rU
= peak area of all-trans-beta carotene from the
Sample solution
rT
= [(peak area of all-trans-alpha carotene ×
•1.0• (RB 1-Dec-2014)) + (peak area of all-transbeta carotene) + (peak area of 9-cis-beta
carotene) + (peak area of 13-cis-beta
carotene × 1.2) + (peak area of 15-cis-beta
carotene × 1.4) + (sum of peak areas of
other cis-isomers of beta carotene)] from the
Sample solution
Acceptance criteria: NLT •95%• (RB 1-Dec-2014) of alltrans-beta carotene in the total carotenoids content
.
.
•• (RB 1-Dec-2014)
.
rT
.
.
IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 0.2%, 2 g of specimen
being used
Delete the following:
•• HEAVY METALS, Method II 〈231〉: NMT 10 ppm• (Official 1.
Dec-2015)
SPECIFIC TESTS
Delete the following:
•• MELTING RANGE OR TEMPERATURE 〈741〉: 176°–182°,
with decomposition• (RB 1-Dec-2014)
.
Change to read:
• ALPHA CAROTENE AND OTHER RELATED COMPOUNDS
Mobile phase, System suitability solution, Standard
solution, Sample solution, and Chromatographic system: Proceed as directed in the test for Content of
Beta Carotene.
Analysis
Sample: Sample solution
•• (RB 1-Dec-2014)
.
Calculate the percentage of alpha carotene and other
individual related compounds relative to total carotenoids in the portion of the Sample taken:
• LOSS ON DRYING 〈731〉
Analysis: Dry under vacuum over phosphorus pentoxide at 40° for 4 h.
Acceptance criteria: NMT 0.2%
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
• USP REFERENCE STANDARDS 〈11〉
USP Beta Carotene RS
(all-E)-1,1′-(3,7,12,16-Tetramethyl1,3,5,7,9,11,13,15,17-octadecanonaene-1,18diyl)bis[2,6,6-trimethylcyclohexene].
USP Beta Carotene System Suitability RS
Result = (rU/rT) × 100
rU
= (peak area of all-trans-alpha carotene ×
•1.0• (RB 1-Dec-2014)) or (peak area response of
other individual related compounds ×
appropriate relative response factor, Table 1)
in the Sample solution
.
2014 The United States Pharmacopeial Convention All Rights Reserved.